Assays for modulators of asparaginyl hydroxylase

ABSTRACT

A method of identifying an agent which modulates hydroxylation of hypoxia inducible factor (HIF), comprises contacting a HIF asparagine hydroxylase and a test substance in the presence of a substrate of the hydroxylase under conditions in which asparagine in the substrate is hydroxylated in the absence of the test substance; and determining hydroxylation of the substrate. Preferably the substrate is a HIF polypeptide comprising HIF-1α, a fragment thereof comprising Asn 803 of HIF-1α or a peptide analogue of HIF-1α or fragment thereof comprising an asparagine equivalent to Asn 803 of HIF-1α and wherein hydroxylation of Asn 803 or of a said equivalent asparagine is determined.

This is a national stage application of International Application No.PCT/GB03/02257, filed May 23, 2003, which claims benefit of priority toGB 0211920.4, filed May 23, 2002.

FIELD OF THE INVENTION

The invention relates to hydroxylases which act on hypoxia induciblefactor (HIF) and which are involved in activation of HIF relatedactivity. The invention also relates to modulators of such hydroxylasesand their use in methods of treatment.

BACKGROUND OF THE INVENTION

Hypoxia in animals activates a broad range of homeostatic responses viainduction of a transcriptional complex termed hypoxia inducible factor(HIF). HIF is a heterodimer of α- and β-subunits, with regulation bydioxygen availability being mediated by post-translational modificationof the α-subunits. In mammalian cells, at least two HIF-α subunitisoforms (HIF-1α and HIF-2α) are regulated by dioxygen levels. EachHIF-α protein contains an internal oxygen dependent degradation domain(ODDD) possessing targeting motifs for proteolytic regulation and aC-terminal transactivation domain (CAD) independently regulated bydioxygen, irrespective of changes in protein abundance, throughinteraction with the CH1 domain of the co-activator p300.

The oxygen dependent degradation of HIF-α by proteolysis is regulated bythe hydroxylation of specific prolyl residues (Pro-402 and Pro-564 inhuman HIF-1α) that mediate recognition of HIF-α by the von Hippel-Lindau(VHL) ubiquitinylation complex, and consequent proteasomal destruction.Combined structural analysis and genetic approaches led to theidentification of three isoforms of human HIF prolyl hydroxylase(PHD1-3, prolyl hydroxylase domain) together with homologues in a rangeof organisms. In vitro analyses, together with sequence and mutationalanalyses identified these as belonging to a sub-family of the Fe(II) and2-oxoglutarate (2-OG) dependent oxygenases. Limiting oxygen availabilityin hypoxia, or direct inhibition of the PHD enzymes by cobaltous ionsand iron chelators, allows HIF-α to escape hydroxylation and recognitionby pVHL, providing insights into the mechanism by which these stimulisuppress HIF-αdegradation and activate the transcriptional cascade.

Previous analyses of the HIF-α CAD have indicated that, as withproteolysis, the action of hypoxia is mimicked by cobaltous ions andiron chelators.

Mass spectrometric and mutational analyses of the HIF-α CAD demonstrateregulatory hydroxylation of a specific asparaginyl residue (Asn-803 inHIF-1α). In the presence of oxygen, hydroxylation at this site preventsinteraction with the p300 CH1 domain, whereas in hypoxia suppression ofthe modification allows interaction with p300 and transcriptionalactivation. Consistent with this model, NMR studies of the human HIF-1αCAD complexed to CH1 indicate that the unmodified Asn-803 is buried atthe interface between the proteins.

SUMMARY OF THE INVENTION

The present inventors have now identified that hydroxylation of Asn-803in HIF-1α is catalysed by Factor Inhibiting HIF (FIH). Hydroxylation ofAsn-803 interferes with interaction of p300 CH1 domain, and thus reducestranscriptional activation mediated by HIF.

In accordance with the present invention, there is provided an assay foridentifying an agent which modulates asparagine hydroxylation of HIF,the method comprising:

-   -   contacting a HIF asparagine hydroxylase and a test substance in        the presence of a substrate of the hydroxylase under conditions        which allow hydroxylation of the substrate in the absence of the        test substance; and    -   determining hydroxylation of the substrate.

The invention also relates to the use of substances identified inaccordance with the assays of the present invention and to the use ofmodulators of the asparagine hydroxylases described herein in thetreatment of a condition or disease associated with altered HIF levelswith respect to healthy or normal levels, or a condition in which it isdesired to alter HIF activity.

DESCRIPTION OF THE SEQUENCES

-   SEQ ID NO: 1 comprises the nucleotide and amino acid sequence for    FIH, HIF asparagine hydroxylase.-   SEQ ID NO: 2 comprises the amino-acid sequence for FIH.-   SEQ ID NO: 3 comprises the amino acid sequence of a modified FIH.-   SEQ ID NOs: 4 to 13 are primers used in the Examples.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to identification of HIF asparaginehydroxylase and provides for the use of such hydroxylases in assaymethods to identify modulators of HIF mediated activity.

In preferred aspects of the invention, a HIF hydroxylase, variant orfragment for use in accordance with the invention has the ability tohydroxylate one or more residues of HIF-1α, preferably asparaginylresidue of HIF and in particular Asn 803 of HIF-1α or a peptide analogueof HIF-1 or fragment thereof incorporating such an asparaginyl.Preferably, a variant of a HIF asparagine hydroxylase in accordance withthe present invention has at least 60% sequence identity with the aminoacid sequence of SEQ ID NO: 2, preferably greater than 70%, morepreferably greater than about 80%, 90% or 95% sequence identity.

The present invention also includes use of active portions, fragments,derivatives and functional mimetics of the polypeptides of theinvention. An “active portion” of a polypeptide means a peptide which isless than said full length polypeptide, but which retains hydroxylaseactivity and in particular maintains HIF asparagine hydroxylaseactivity. Such an active fragment may be included as part of a fusionprotein, e.g. including a binding portion for a different i.e.heterologous ligand.

Typically, polypeptides with more than about 60% identity preferably atleast 70%, at least 80% or at least 90% and particularly preferably atleast 95% or at least 99% identity, with the amino acid sequence of SEQID NO: 2, are considered as variants of the proteins. Such variants mayinclude allelic variants and the deletion, modification or addition ofsingle amino acids or groups of amino acids within the protein sequence,as long as the peptide retains asparagine hydroxylase activity.Preferably a variant of SEQ ID NO: 2 will have the same domain structureas FIH, i.e. an eight strand β barrel jelly roll.

Amino acid substitutions may be made, for example from 1, 2 or 3 to 10,20 or 30 substitutions. Conservative substitutions may be made, forexample according to the following Table. Amino acids in the same blockin the second column and preferably in the same line in the third columnmay be substituted for each other.

ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

Variant polypeptides within the scope of the invention may be generatedby any suitable method, for example by gene shuffling (molecularbreeding) techniques.

Shorter polypeptide sequences are within the scope of the invention. Forexample, a peptide fragment of at least 20 amino acids or up to 50, 60,70, 80, 100, 150 or 200 amino acids in length is considered to fallwithin the scope of the invention. In particular, but not exclusively,this aspect of the invention encompasses the situation when the proteinis a fragment of the complete protein sequence and may represent acatalytic region, capable of hydroxylating Asn 803 of HIF-α. Suchfragments can be used to construct chimeric molecules.

Polypeptides of the invention may be chemically modified, e.g.post-translationally modified. For example, they may be glycosylated orcomprise modified amino acid residues. They may also be modified by theaddition of histidine residues to assist their purification or by theaddition of a nuclear localisation sequence to promote translocation tothe nucleus or by post translational modification includinghydroxylation or phosphorylation. Such modified polypeptides fall withinthe scope of the term “polypeptide” of the invention.

The polypeptides of the invention may be used in assays for asparaginylhydroxylase activity on substrates such as HIF. The polypeptides mayalso be used for hydroxylation of suitable substrates and in particularasparaginyl hydroxylation of such substrates, in particular where it isdesired to have specific hydroxylation of asparagine with little or nohydroxylation of other residues such as aspartic acid. A variant or anactive fragment of a HIF asparagine hydroxylase of the invention maytypically be identified by monitoring for hydroxylase activity asdescribed in more detail below.

Such HIF asparagine hydroxylases may be a eukaryotic polypeptide,preferably a mammalian polypeptide, more preferably a human polypeptidesuch as that of SEQ ID NO: 2.

A HIF asparagine hydroxylase preferably contains a jelly roll (doublestranded beta helix/β barrel jelly roll) structure consisting of aminimum of eight strands. Typically, the jelly roll structure may haveeight strands, although an insert is preferably present between βstrands 4 and 5 of the jelly roll. Preferred HIF hydroxylases containthe sequence;HXD/E[X]_(n)Hwhere X is any amino acid and n is between 1 and 200, 20 and 150 or 30and 100 amino acids, for example 10, 20, 30, 40, 50, 60, 70, 80, 90 or100 amino acids.

Nucleotides according to the invention encoding HIF asparaginylhydroxylase have utility in production of the proteins according to theinvention, which may take place in vitro, in vivo or ex vivo. Thenucleotides may be involved in recombinant protein synthesis or indeedas therapeutic agents in their own right, utilised in gene therapytechniques. Nucleotides complementary to those encoding HIF asparaginehydroxylase, or antisense sequences, or interfering RNA may also be usedin gene therapy.

A polynucleotide of the invention can hybridize to the coding sequenceor the complement of the coding sequence of SEQ ID NO: 1 at a levelsignificantly above background. Background hybridization may occur, forexample, because of other cDNAs present in a cDNA library. The signallevel generated by the interaction between a polynucleotide of theinvention and the coding sequence or complement of the coding sequenceof SEQ ID NO: 1 is typically at least 10 fold, preferably at least 100fold, as intense as interactions between other polynucleotides and thecoding sequence of SEQ ID NO: 1. The intensity of interaction may bemeasured, for example, by radiolabelling the probe, e.g. with ³²PSelective hybridisation may typically be achieved using conditions ofmedium to high stringency. However, such hybridisation may be carriedout under any suitable conditions known in the art (see Sambrook et al,1989. For example, if high stringency is required suitable conditionsinclude from 0.1 to 0.2×SSC at 60° C. up to 65° C. If lower stringencyis required suitable conditions include 2×SSC at 60° C.

The coding sequence of SEQ ID NO: 1 may be modified by nucleotidesubstitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100substitutions. The polynucleotide of SEQ ID NO: 1 may alternatively oradditionally be modified by one or more insertions and/or deletionsand/or by an extension at either or both ends. A polynucleotide mayinclude one or more introns, for example may comprise genomic DNA. Themodified polynucleotide generally encodes a polypeptide which hasasparagine hydroxylase activity. Degenerate substitutions may be madeand/or substitutions may be made which would result in a conservativeamino acid substitution when the modified sequence is translated, forexample as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to thecomplement of the DNA coding sequence of SEQ ID NO: 1 will generallyhave at least 50%, at least 57%, at least 60%, at least 70%, at least80%, at least 88%, at least 90%, at least 95%, at least 98% or at least99% sequence identity to the coding sequence of SEQ ID NO: 1 over aregion of at least 20, preferably at least 30, for instance at least 40,at least 60, more preferably at least 100 contiguous nucleotides or mostpreferably over the full length of SEQ ID NO: 1. Preferably thenucleotide sequence encodes a polypeptide which has the same domainstructure as FIH.

For example the UWGCG Package provides the BESTFIT program which can beused to calculate homology (for example used on its default settings)(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (typically on their default settings), for example asdescribed in Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al(1990) J. Mol. Biol. 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Centre for Biotechnology Information (www.ncbi.nlm.mh.gov/).This algorithm involves first identifying high scoring sequence pair(HSPs) by identifying short words of length W in the query sequence thateither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighbourhood word score threshold (Altschul et al,1990). These initial neighbourhood word hits act as seeds for initiatingsearches to find HSPs containing them. The word hits are extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Extensions for the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a word length (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787 and Altschul and Gish (1996) MethodsEnzymol. 266: 460-480. One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a sequence isconsidered similar to another sequence if the smallest sum probabilityin comparison of the first sequence to the second sequence is less thanabout 1, preferably less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Any combination of the above mentioned degrees of sequence identity andminimum sizes may be used to define polynucleotides of the invention,with the more stringent combinations (i.e. higher sequence identity overlonger lengths) being preferred. Thus, for example a polynucleotidewhich has at least 90% sequence identity over 25, preferably over 30nucleotides forms one aspect of the invention, as does a polynucleotidewhich has at least 95% sequence identity over 40 nucleotides.

The present invention also includes expression vectors that comprisenucleotide sequences encoding the proteins of the invention. Suchexpression vectors are routinely constructed in the art of molecularbiology and may for example involve the use of plasmid DNA andappropriate initiators, promoters, enhancers and other elements, such asfor example polyadenylation signals which may be necessary, and whichare positioned in the correct orientation, in order to allow for proteinexpression. Other suitable vectors would be apparent to persons skilledin the art. By way of further example in this regard we refer toSambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into thevectors described above in an antisense orientation in order to providefor the production of antisense RNA. Antisense RNA or other antisiensepolynucleotides may also be produced by synthetic means. Such antisensepolynucleotides may be used as test compounds in the assays of theinvention or may be useful in a method of treatment of the human oranimal body by therapy.

Polynucleotides of the invention may also be used to design doublestranded RNAs for use in RNA interference. Such RNA comprises shortstretches of double stranded RNA having the same sequence as a targetmRNA. Such sequences can be used to inhibit translation of the mRNA.Alternatively, small fragments of the gene encoding a HIF asparaginehydroxylase may be provided, cloned back to back in a plasmid.Expression leads to production of the desired double stranded RNA. Suchshort interfering RNA (siRNA) may be used for example to reduce orinhibit expression of a HIF hydroxylase of the invention, in assays orin a method of therapy. The invention also relates to such siRNAs. SuchsiRNAs may be designed to inhibit groups of HIF hydroxylases of theinvention by basing their sequences on regions of conserved sequence inthe encoding genes of the hydroxylases. Alternatively, the siRNAs may bemade specific to a particular HIF hydroxylase by choosing a sequenceunique to the encoding gene of the particular hydroxylase gene to beinhibited.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence which is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A regulatorysequence, such as a promoter, “operably linked” to a coding sequence ispositioned in such a way that expression of the coding sequence isachieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors providedwith a origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.The vector may be an artificial chromosome. The vectors may contain oneor more selectable marker genes, for example an ampicillin resistancegene in the case of a bacterial plasmid or a resistance gene for afungal vector. Vectors may be used in vitro, for example for theproduction of DNA or RNA or used to transfect or transform a host cell,for example, a mammalian host cell. The vectors may also be adapted tobe used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to becompatible with the host cell for which expression is designed. Forexample, yeast promoters include S. cerevisiae GAL4 and ADH promoters,S. pombe nmt1 and adh promoter. Mammalian promoters include themetallothionein promoter which can be induced in response to heavymetals such as cadmium. Viral promoters such as the SV40 large T antigenpromoter or adenovirus promoters may also be used. An IRES promoter mayalso be used. All these promoters are readily available in the art.

Mammalian promoters, such as β-actin promoters, may be used.Tissue-specific promoters are especially preferred. Inducible promotersare also preferred. Promoters inducible by hypoxic conditions may, forexample, be employed. Viral promoters may also be used, for example theMoloney murine leukaemia virus long terminal repeat (MMLV LTR), the roussarcoma virus (RSV) LTR promoter, the SV40 promoter, the humancytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such asthe HSV IE promoters), or HPV promoters, particularly the HPV upstreamregulatory region (URR). Viral promoters are readily available in theart.

The vector may further include sequences flanking the polynucleotidegiving rise to polynucleotides which comprise sequences homologous toeukaryotic genomic sequences, preferably mammalian genomic sequences, orviral genomic sequences. This will allow the introduction of thepolynucleotides of the invention into the genome of eukaryotic cells orviruses by homologous recombination. In particular, a plasmid vectorcomprising the expression cassette flanked by viral sequences can beused to prepare a viral vector suitable for delivering thepolynucleotides of the invention to a mammalian cell. Homologousrecombination may also be used to disrupt or mutate endogenous sequencesin cells encoding HIF asparagine hydroxylases. Other examples ofsuitable viral vectors include herpes simplex viral vectors andretroviruses, including lentiviruses, adenoviruses, adeno-associatedviruses and HPV viruses. Gene transfer techniques using these virusesare known to those skilled in the art. Retrovirus vectors for examplemay be used to stably integrate the polynucleotide giving rise to thepolynucleotide into the host genome. Replication-defective adenovirusvectors by contrast remain episomal and therefore allow transientexpression.

The invention also includes cells that have been modified to express aHIF asparagine hydroxylase of the invention.

Assays

Our data shows that hydroxylation of HIF-α CAD is mediated by anasparagine hydroxylase enzyme. The enzyme responsible is referred to asHIF asparagine hydroxylase and includes FIH. The action of HIFasparagine hydroxylases, and in particular human HIF hydroxylases,represent a novel target for the control of HIFα. By blocking HIFasparagine hydroxylase activity, this will reduce hydroxylation of HIF-αand thus lead to an increase in the interaction with p300 and inparticular the CH1 domain and thus transcriptional activation. This inturn will lead to the activation of systemic local defences againsthypoxia or ischaemia that may include the promotion of angiogenesis,erythropoiesis, energy metabolism, inflammation, vasomotor function, andwill also affect apoptotic/proliferative responses.

We describe below in more detail a number of different assays that maybe carried out to identify modulators of HIF hydroxylase activity and inparticular of asparagine hydroxylase activity, or which affectregulation of HIF-α interaction with p300 in a cell and hence whichaffect HIF mediated activity. Some of these assays utilise HIFpolypeptides, and in particular HIF asparagine hydroxylases inaccordance with the present invention. Typically, the assays may utilisea human HIF asparagine hydroxylase such as FIH or a fragment or variantof a human HIF asparagine hydroxylase. Non-human HIF hydroxylases mayalso be used. These components are described in more detail below. Eachof these components, where required may be provided either in purifiedor unpurified form, for example, as cellular extracts or by purificationof the relevant component from such extracts. Alternatively, therelevant component can be expressed using recombinant expressiontechniques and purified for use in the assay. Alternatively, thecomponents may be expressed recombinantly in a cell for use in cellbased assays.

Typically, a polynucleotide encoding the relevant component is providedwithin an expression vector. Such expression vectors are routinelyconstructed in the art and may for example involve the use of plasmidDNA and appropriate initiators, promoters, enhancers and other elements,such as for example polyadenylation signals which may be necessary andwhich are positioned in the correct orientation in order to allow fullprotein expression. Suitable vectors would be very readily apparent tothose of skill in the art, such as those described in more detail hereinwith reference to the HIF hydroxylases. Promoter sequences may beinducible or constitutive promoters depending on the selected assayformat. The promoter may be tissue specific. Examples of promoters andother flanking sequences for use in the expression vectors are describedin more detail herein with reference to the HIF hydroxylases of theinvention and in particular to the human HIF hydroxylases of theinvention.

HIF Polypeptides and Peptide Analogues

The assays of the present invention may use a substrate of a HIFasparagine hydroxylase and in particular an asparagine containingsubstrate of the enzyme. In particular, such substrates may be used inassays to monitor for the activity of a modulator of HIF asparaginehydroxylase activity. The substrate may be a HIF polypeptide or peptideanalogue thereof. Typically, a HIF polypeptide will be used as thesubstrate.

Any suitable substrate in which an asparagine residue is hydroxylated bya HIF hydroxylase of SEQ ID NO: 2 may be used. In preferred embodiments:of the invention, such a substrate is a HIF polypeptide such as a HIF-1αor HIF-2α subunit protein or fragment of either or peptide analogue ofthe subunit or fragment. Preferably, the HIF-α peptide conveys an oxygenregulated response. Preferably, the HIF-α peptide has a CAD domain andis capable of oxygen regulated interaction with p300 and downstreamtranscriptional activation. Preferably, such HIF-αpeptides are capableof interacting with the p300 CH1 domain. Preferably, such HIFpolypeptides, fragments or peptide analogues incorporate an asparagineresidue equivalent to Asn 803 defined with reference to HIF-1α. Theasparagine equivalent to Asn 803 of HIF-1α may be determined by aligningthe HIF variant, fragment or analogue to the sequence of HIF-1α toobtain the best sequence alignment and identifying thereby theasparagine equivalent to Asn 803 of HIF-1α. A HIF polypeptide may be ofeukaryotic origin, in particular a human or other mammalian, HIF-αsubunit protein or fragment thereof. Alternatively, the polypeptide maybe of C. elegans origin. In those assays which monitor for hydroxylationof HIF-α through its interaction with p300, the HIF polypeptide has theability to bind to a wild type full length p300 protein or a fragmentthereof comprising the CH1 domain. Preferably, such binding is able, ina hypoxic cellular environment, to activate transcription.

A number of HIFα subunit proteins have been cloned. These includeHIF-1α, the sequence of which is available as Genbank accession numberU22431, HIF-2α, available as Genbank accession number U81984 and HIF-3α,available as Genbank accession numbers AC007193 and AC079154. These areall human HIF α subunit proteins and all may be used in the invention.HIF-α subunit proteins from other species, including murine HIF-1α(accession numbers AF003695, U59496 and X95580), rat HIF-1α (accessionnumber Y09507), murine HIF-2α (accession numbers U81983 and D89787) andmurine HIF-3α (accession number AF060194) may also be used in theinvention.

One HIF-α protein of particular interest is the C. elegans HIF-α subunitprotein. The C. elegans system may be used in assays of the presentinvention.

There are a number of common structural features found in the two HIF-αsubunit proteins identified to date. Some of these features areidentified in O'Rourke et al (1999, J. Biol. Chem., 274; 2060-2071) andmay be involved in the trans-activation functions of the HIF-α subunitproteins. One or more of these common structural features are preferredfeatures of the HIF polypeptides.

Variants of the above HIF-α subunits may be used, such as syntheticvariants which have at least 45% amino acid identity to a naturallyoccurring HIF-α subunit (particularly to a human HIF-α subunit such as,for example HIF-1α), preferably at least 50%, 60%, 70%, 80%, 90%, 95% or98% identity. Such variants may include substitutions or modificationsas described above with respect to HIF hydroxylases. Amino acid activitymay also be calculated as described above with reference to HIFhydroxylases.

HIF fragments may also include non-peptidyl functionalities and may beoptimised for assay purposes such that the level of identity is lowered.Such functionalities may be covalently bound such as sugars ornon-covalently bound such as metal ions.

HIFα polypeptides as described herein may be fragments of the HIF-αsubunit protein or variants as described above, provided that saidfragments retain the ability to interact with a wild-type p300 CH1domain. When using proteinogenic amino acid residues, such fragments aredesirably at least 20, preferably at least 40, 50, 75, 100, 200, 250 or400 amino acids in size. Desirably, such fragments include asparagine803.

Cell based assays of the present invention may involve upregulation ofan endogenous HIF-α or expression of a HIF-α by recombinant techniquesand in particular of HIF-1α.

Assay Methods

The present invention provides an assay method for identifying an agentwhich modulates asparagine hydroxylation of hypoxia inducible factor.The method comprises contacting a HIF asparagine hydroxylase and a testsubstance, such as a potential inhibitor, in the presence of a substrateof the hydroxylase under conditions in which asparagine hydroxylationoccurs in the absence of the test substance and determining the extentof asparagine hydroxylation of the substrate. Alternatively, the assaymay be used to detect substances that increase the activity of the HIFasparagine hydroxylase by assaying for increases in activity.

In the Experiments described herein, FIH has been found to hydroxylateHIF-α at an asparagine residue within the CAD domain. This hydroxylationmediates p300 binding and in particular, reduces p300 binding. Suchbinding leads to transcriptional activation. This interaction andactivation may also be used as the basis for an assay of the invention.

Such assays of the present invention may be used to identify inhibitorsof HIF asparagine hydroxylase activity and are thus preferably carriedout under conditions under which asparagine hydroxylation would takeplace in the absence of the test substance. In the alternative, theassays may be used to look for promoters of asparagine hydroxylaseactivity, for example, by looking for increased hydroxylation ofasparagine substrates compared to assays carried out in the absence of atest substance. The assays may also be carried out under conditions inwhich hydroxylation is reduced or absent, such as under hypoxicconditions and the presence of or increased hydroxylation could bemonitored under such conditions. The assays of the invention may also beused to identify inhibitors or activitators which are specific for HIFasparagine hydroxylases and which do not have activity or are lessactive with other hydroxylases, for example, such as HIF prolylhydroxylases or other asparagine/aspartamic acid hydroxylases.

The present invention also provides an assay method for theidentification of other HIF asparagine hydroxylases. The methodtypically comprises providing a test polypeptide; bringing into contacta HIF polypeptide and the test polypeptide under conditions in which theHIF polypeptide is hydroxylated at an asparagine residue by HIFhydroxylase and determining whether or not the HIF polypeptide ishydroxylated at the asparagine residue.

Further, other proteins are clearly related to FIH by sequence includingactive site residues and specifically those residues that are involvedin binding the asparagine residue that is hydroxylated in HIF. Theseinclude those represented by SWALL database codes: Q9NWJ5, Q8TB10,Q9Y4E2, O95712, Q9H8B1, Q9NWT6 in Homo sapiens, and Q91W88 and Q9ER15 inMus musculus (FIH=Q969Q7). Some or all of these have already beenassociated with transcription (Clissold P M, Ponting C P, JmjC: cupinmetalloenzyme-like domains in jumonji, hairless and phospholipaseA(2)beta TRENDS BIOCHEM SCI 26 (1): 7-9 January 2001), thus specificmodulation of FIH activity alone or the modulation of FIH activitytogether with at least one of the related enzymes is of interest.

The present invention also provides an assay method for identifyingalternative substrates of the HIF asparagine hydroxylase of theinvention. Such assay method typically comprises contacting a testpolypeptide with a HIF asparagine hydroxylase of the invention underconditions in which HIF would normally be hydroxylated at asparagine bythe hydroxylase and determining whether the test polypeptide ishydroxylated at asparagine.

Methods for Monitoring Modulation

The precise format of any of the screening or assay methods of thepresent invention may be varied by those of skill in the art usingroutine skill and knowledge. The skilled person is well aware of theneed to additionally employ appropriate controlled experiments. Theassays of the present invention may involve monitoring for asparaginehydroxylation of a suitable substrate, monitoring for the utilisation ofsubstrates and co-substrates, monitoring for the production of theexpected products between the enzyme and its substrate. Assay methods ofthe present invention may also involve screening for the directinteraction between components in the system. Alternatively, assays maybe carried out which monitor for downstream effects such as binding ofHIF by p300 and downstream effects mediated by HIF such as HIF mediatedtranscription using suitable reporter constructs or by monitoring forthe upregulation of genes or alterations in the expression patterns ofgenes know to be regulated directly or indirectly by HIF.

Various methods for determining hydroxylation are known in the art andare described and exemplified herein. Any suitable method may be usedfor determining activity of the HIF hydroxylase such as by substrate orco-substrate utilization, product appearance such as peptidehydroxylation or down-stream effects mediated by hydroxylated ornon-hydroxylated products.

Our finding that the Asn 803 residue of HIF-1α is hydroxylated by anasparagine hydroxylase provides the basis for assay methods designed toscreen for inhibitors or promoters of this process. Any suitable methodmay be used to monitor for hydroxylation of HIF-1α or a HIF polypeptideor analogue thereof. Assays may be carried out to monitor directly forhydroxylation of the relevant asparagine residue or another position.Alternatively, assays may be carried out to monitor for depletion ofco-factors and co-substrates. Alternatively, such assays may monitor thedownstream effects of hydroxylation of HIF or indeed inhibition ofhydroxylation of HIF, for example, by monitoring the interaction betweenHIF and p300 or HIF mediated transcription. Alternatively, reporter geneconstructs driven by HIF regulated promoters may be used. Assays arealso provided for the identification of enhancers of the activity of theHIF asparagine hydroxylase. Such enhancers may be used to reduce HIFαactivity.

In one embodiment, a suitable substrate of the HIF asparaginehydroxylase is provided. This may be HIF-α or a fragment thereof whichincludes a CAD domain or which includes a residue equivalent to Asn 803of HIF-1α. The substrate may not be initially hydroxylated at the Asn803 position. This may be achieved by providing synthetic polypeptidesubstrates, or by producing HIF-α polypeptides in bacterial cells,insect cells or mammalian cells or in in vitro transcription andtranslation systems. Alternatively, assays may be carried out over aselected time course such that the substrate is produced during thecourse of the assay, initially in un-hydroxylated form.

The substrate, enzyme and potential inhibitor compound may be incubatedtogether under conditions which, in the absence of inhibitor provide forhydroxylation of Asn 803, and the effect of the inhibitor may bedetermined by determining hydroxylation of the substrate. This may beaccomplished by any suitable means. Small polypeptide substrates may berecovered and subject to physical analysis, such as mass spectrometry orchromatography, or to functional analysis, such as the ability to bindto p300 (or displace a reporter molecule from p300). Such methods areknown as such in the art and may be practiced using routine skill andknowledge. Determination may be quantitative or qualitative. In bothcases, but particularly in the latter, qualitative determination may becarried out in comparison to a suitable control, e.g. a substrateincubated without the potential inhibitor.

Inhibitor compounds which are identified in this manner may be recoveredand formulated as pharmaceutical compositions.

Another assay of the invention is for a promoter of asparaginehydroxylation of HIF-α subunits. Typically, a HIF-α subunit or portionthereof is prepared as described above, and incubated under hypoxicconditions. By “hypoxic”, it is meant less than 5%, preferably less than3%, more preferably less than 1%, end preferably less than 0.5%, such asless than 0.1% O₂. The HIF-α subunit is incubated with a cell extractwhich includes the HIF asparagine hydroxylase as described above,optionally further in the presence of a source of ferrous (FeII) ions,and/or other co-factors. A suitable concentration of ferrous ions is inthe range of from 1 to 500 μM, such as from 25 to 250 μM and inparticular from 50-200 μM. Ferrous ions may be supplied in the form offerrous chloride, ferrous sulphate, and the like.

In this embodiment of the invention, the substrate will be incubated inthe presence of a potential hydroxylation promoting agent, and theeffect of the agent determined, by determining the hydroxylation of theAsn 803. As with the assay of the other aspect of the inventiondescribed above, determination may be quantitative or qualitative, andin either case determined relative to a suitable control.

The interaction between HIF and p300 is mediated by hydroxylation ofHIF. Assays in accordance with the present invention may involvemonitoring for the interaction between p300 and HIF. In particular, theinteraction can be monitored for example by the use of fluorescencepolarisation, surface plasmon resonance or mass spectrometric analysis.In the first instance, the fluorescence polarisation of a dye attachedto the test polypeptide changes when interaction with p300 occurs, aninteraction which is itself dependent on the hydroxylation state of thetest polypeptide. In the second instance, a test polypeptide may beimmobilised on a chip constructed such that binding events may bedetected by a change in force exerted on the chip. “Native” or “softionisation” mass spectrometry can be used as an assay for hydroxylaseactivity; thus interactions between HIFα polypeptide, or fragmentthereof containing the C-terminal transactivation domain, and p300 areobserved by mass spectrometry, whereas upon hydroxylation, thisinteraction may be reduced or abrogated. Transcription and expression ofgenes known to be upregulated or down regulated by the presence of HIFcan be monitored. In particular, upregulation of HIF regulated geneswould demonstrate inhibition of asparagine hydroxylation whereas downregulation would suggest enhancement or promotion of asparaginehydroxylation.

In alternative embodiments, reporter constructs may be provided in whichpromoters mediated by HIF are provided operably linked to a reportergene. Any suitable reporter gene could be used, such as for exampleenzymes which may then be used in colorometric, fluorometric,fluorescence resonance or spectrometric assays.

HIF asparagine hydroxlase is a 2OG dependent oxygenase.

In the assay methods described herein, typically the HIF asparaginehydroxylase and the substrate of the hydroxylase are contacted in thepresence of a co-substrate, such as 2-oxoglutarate (2OG) or dioxygen.The hydroxylase activity of the HIF hydroxylase may be determined bydetermining the turnover of the co-substrate. This may be achieved bydetermining the presence and/or amount of reaction products, such ashydroxylated substrate or succinic acid. The amount of product may bedetermined relative to the amount of substrate. Typically, in suchembodiments the substrate may be an HIF-α polypeptide and, for example,the product measured may be hydroxylated HIF-α polypeptide. For example,the extent of hydroxylation may be determined by measuring the amount ofhydroxylated HIFα polypeptide, succinate or carbon dioxide generated inthe reaction, or by measuring the depletion of 2OG or dioxygen. Methodsfor monitoring each of these are known in the scientific literature.

HIFα asparagme hydroxylase activity may be determined by determining theturnover of said 2OG to succinate and CO₂, as described in Myllyhaiju J.et al EMBO J. 16 (6): 1173-1180 (1991) or as in Cunliffe C. J. et alBiochem. J. 240 617-619 (1986), or other suitable assays for CO₂,bicarbonate or succinate production.

Unused 2OG may be derivatised by chemical reagents, exemplified by butnot limited to hydrazine derivatives and ortho-phenylene diaminederivatives, to give indicative chromophores or fluorophores that can bequantified and used to indicate the extent of hydroxylation of the testpolypeptide. Dissolved oxygen electrodes, exemplified by but not limitedto a “Clarke-type” electrode or an electrode that uses fluorescencequenching, may be used to follow the consumption of oxygen in an assaymixture, which can then be used to indicate the extent of hydroxylationof the test polypeptide in an analogous manner to the above.

Alternatively, the end-point determination may be based on conversion ofHIFα or peptide fragments (including synthetic and recombinant peptides)derived from HIFα into detectable products. Peptides may be modified tofacilitate the assays so that they can be rapidly carried out and may besuitable for high throughput screening.

For example, reverse phase HPLC (C-4 octadecylsilane column), asexemplified herein, may be used to separate starting synthetic peptidesubstrates for HIF hydroxylase from the asparagine hydroxylatedproducts, as the latter have a shorter retention time in the column.Modifications of this assay or alternative assays for HIF hydroxylaseactivity may employ, for example, mass spectrometric, spectroscopic,and/or fluorescence techniques as are well known in the art(Masimirembwa C. et al Combinatorial Chemistry & High ThroughputScreening (2001) 4 (3) 245-263, Owicki J. (2000) J. Biomol. Screen. 5(5) 297-305, Gershkovich A et al (1996) J. Biochem. & Biophys. Meths. 33(3) 135-162, Kraaft G. et al (1994) Meths. Enzymol. 241 70-86).Fluorescent techniques may employ versions of the substrate modified insuch as way as to carry out or optimise spectroscopic or fluorescenceassays.

For example, HIFα polypeptide may be immobilised e.g. on a bead orplate, and hydroxylation of the appropriate residue detected using anantibody or other binding molecule which binds the CAD binding domain ofHIFα with a different affinity when an asparagine 803 is hydroxylatedfrom when the residue is not hydroxylated. Such antibodies may beobtained by means of standard techniques which are well known in theart, e.g. using a hydroxylated HIFα peptide.

Binding of a molecule which discriminates between the hydroxylated andnon-hydroxylated form of a HIFα polypeptide may be assessed using anytechnique available to those skilled in the art, which may involvedetermination of the presence of a suitable label.

Assay methods of the present invention may also take the form of an invivo assay. The in vivo assay may be performed in a cell line such as ayeast strain in which the relevant polypeptides or peptides areexpressed from one or more vectors introduced into the cell.

In Vivo Assays

The assays may be carried out using cell based, organ based or wholeanimal assays conducted in vivo. Such assays may utilize the endogenousexpression of the HIF hydroxylase nucleotides and/or polypeptides. Inother forms of the invention, upregulation of specific endogenous HIFhydroxylases may be achieved by stimulators of the expression thereof.Such stimulators may be growth factors or chemicals that upregulatespecific HIF asparagine hydroxylases. In another form of the invention,nucleotide constructs may be introduced into cells or transgenic animalsto increase production of one or more specific HIF asparaginehydroxylases. Alternatively nucleotide constructs may be introduced intocells so as reduce or abrogate expression of one or more specific HIFhydroxylases. Appropriate methods that include but are not limited tohomologous recombination, antisense expression, ribozyme expression andRNA interference are outlined herein and known by those skilled in theart.

Tissue culture cells, organs, animals and other biological systems,obtained by the aforementioned forms of the invention, may be used toprovide a further source of a HIF hydroxylase, or may be used for theassay, or especially comparative assay, of the activity of testsubstances may inhibit, augment, block or otherwise modulate theactivity of specific HIF hydroxylases.

The activity of the HIF hydroxylases may be assayed by any of theaforementioned methods or by cell, tissue, or other assays conducted invivo that measure the effects of altered activity of the HIFhydroxylases.

HIF complexed with p300 activate hypoxia response elements that arefound in the promoters and/or enhancers of endogenous genes that areregulated by the said HIF complexes. Such hypoxia response elements mayalso be isolated and operationally linked to reporter genes so as toassay the activity of the HIF complex through detection and/orquantitation of the reporter gene or its product. Therefore in a furtherform of the invention the activity of a HIF-α polypeptide that isregulated by HIF asparagine hydroxylase will be assayed by measuring theeffects of the HIF complex on the expression of an endogenous gene orreporter gene that is functionally linked to a HIF binding hypoxiaresponse element. Examples of endogenous genes that are regulated inthis way are to be found in the role of the aryl hydrocarbon nucleartranslocator (ARNT) in hypoxic induction of gene expression, see forexample, Studies in ARNT-deficient cells. S. M. Wood, J. M. Gleadle, C.W. Pugh, O. Hankinson, P. J. Ratcliffe. Journal of Biological Chemistry271 (1996) 15117-15123, and Hypoxia inducible expression oftumor-associated carbonic anyhydrases, C. C. Wykoff, N. J. P. Beasley,K. J. Turner, J. Pastorek, A. Sibtain. G. D. Wilson, H. Turley, K.Talks, P. H. Maxwell, C. W. Pugh, P. J. Ratcliffe, A. L. Harris. CancerResearch 60 (2000) 7075-7083. Examples include but are not limited toglucose transporter isoform 1, phosphoglycerate kinase-1, carbonanhydrase isoform 9, vascular endothelial growth factor. Each of saidgenes contains one or hypoxia response elements that may be isolated andoperationally linked as single or multiple copies to a reporter gene forthe measurement of activity of a HIF-α polypeptide that varies inaccordance with the activity of a HIF hydroxylase.

The activity of genes or gene products that are regulated by a HIF-αpolypeptide in accordance with the activity of a HIF hydroxylase affectscellular, organ, and animal physiology in a manner that provide furtheraspects of the invention. Thus a farther embodiment of the inventionprovides for assays that utilise a specific functional response that isregulated in accordance with the activity of a HIF-α polypeptide inaccordance with the activity of a HIF hydroxylase. Such responsesinclude the uptake rate of glucose or glucose analogues that are notmetabolized, the ingrowth of blood vessels by angiogenesis, the activityof a carbonic anhydrase enzyme. It is recognised that many otherresponses that operate at a cellular or systemic level are controlled bythe activity of a HIF-α polypeptide in accordance with the activity of aHIF hydroxylase and may be utilized as assays of the said HIFhydroxylase activity in further aspects of the invention.

A HIF-α polypeptide that is a substrate for a HIF hydroxylase may befused to a further polypeptide so as to cause the activity of the saidHIP hydroxylase to regulate the activity of the fusion peptide.Accordingly a further form of the invention provides for the assay ofthe activity of a fusion polypeptide. In the preferred form such afusion polypeptide may contain the whole of part of a HIF-α polypeptide,particularly including Asn 803, or the CAD domain. The Gal4 DNA bindingdomain including the amino acids 1-143 together with the Gal bindingupstream activating sequence (UAS) is an example of such a transcriptionfactor and cognate DNA response element whose operation can be assayedby those skilled in the art.

Test Compounds

Compounds which may be screened using the assay methods described hereinmay be natural or synthetic chemical compounds used in drug screeningprogrammes. Extracts of plants, microbes or other organisms, whichcontain several characterised or uncharacterised components may also beused.

Combinatorial library technology (including solid phase synthesis andparallel synthesis methodologies) provides an efficient way of testing apotentially vast number of different substances for ability to modulatean interaction. Such libraries and their use are known in the art, forall manner of natural products, small molecules and peptides, amongothers. The use of peptide libraries may be preferred in certaincircumstances.

Potential inhibitor compounds may be polypeptides, small molecules suchas molecules from commercially available combinatorial libraries, or thelike. Small molecule compounds which may be used include 2-oxoglutarateanalogues, or HIF-α analogues, or those that incorporate features ofboth 2-oxoglutarate and affect HIF-α, which inhibit the action of theenzyme. Thus the invention provides the use of a compound which acts asan asparagine hydroxylase inhibitor, for the manufacture of a medicamentfor the treatment of a condition in a patient which requires thepromotion of cell growth, such as angiogenesis. The invention alsoprovides a method of treatment of a patient suffering from a conditionwhich is treatable by promoting cell growth, which comprisesadministering to said patient an effective amount of a HIF asparaginehydroxylase inhibitor.

Potential promoting agents may be screened from a wide variety ofsources, particularly from libraries of small compounds which arecommercially available. Oxygen-containing compounds may be included incandidate compounds to be screened, for example 2-oxoglutarateanalogues.

A test compound which increases, potentiates, stimulates, disrupts,reduces, interferes with or wholly or partially abolishes asparaginehydroxylation of HIF-α polypeptide and which may thereby modulate HIFactivity, may be identified and/or obtained using the assay methodsdescribed herein.

Agents which increase or potentiate asparagine hydroxylation, may beidentified and/or obtained under conditions which, in the absence of apositively-testing agent, limit or prevent hydroxylation. Such agentsmay be used to potentiate, increase, enhance or stimulate the functionof a HIF asparagine hydroxylase, and may have an effect on cells underhypoxic conditions such as those found in tumours, in which the lack ofhydroxylation leads to the accumulation of HIFα and the concomitantpromotion of angiogenesis and other growth promoting events.

Methods of determining the presence of, and optionally quantifying theamount of HIF asparagine hydroxylase in a test sample may have adiagnostic or prognostic purpose, e.g. in the diagnosis or prognosis ofany medical condition discussed herein (e.g. a proliferative disordersuch as cancer) or in the evaluation of a therapy to treat such acondition.

In various aspects, the present invention provides an agent or compoundidentified by a screening method of the invention to be a modulator ofHIFα asparagine hydroxylation e.g. a substance which inhibits orreduces, increases or potentiates the asparagine hydroxylase activity ofa HIF hydroxylase.

Following identification of a modulator, the substance may be purifiedand/or investigated further (e.g. modified) and/or manufactured. Amodulator may be used to obtain peptidyl or non-peptidyl mimetics, e.g.by methods well known to those skilled in the art and discussed herein.A modulator may be modified, for example to increase selectively, asdescribed herein. It may be used in a therapeutic context as discussedbelow.

Selectivity

It may also be advantageous to modulate HIF asparagine hydroxylaseselectively, as a single target, or in selected hydroxylase groups aswell as an entire family. Agents which modulate HIF asparaginehydroxylase activity are therefore preferably specific i.e. they have anincreased or enhanced effect on a HIF asparagine hydroxylase relative toother 2OG dependent oxygenases.

Assay methods as described herein may therefore further comprisecontacting the test compound with one or more 2OG dependent oxygenasesunder conditions in which said 2OG dependent oxygenases are normallyactive and determining activity of said oxygenases. A difference inactivity in the presence relative to the absence of test compound isindicative of the test compound modulating the activity of the one ormore 2OG dependent oxygenases.

A test compound which provides increased or enhanced modulation of a HIFasparagine hydroxylase, relative to the one or more 2OG dependentoxygenases shows selectivity or specificity for the HIF hydroxylase.

2OG dependent oxygenases may include for example, clavaminte synthase,deacetoxycephalosporin C synthase, collagen-prolyl-4-hydroxylase,collagen prolyl-3-hydroxylase, lysyl hydroxylase, aspartyl hydroxylase,phytanoyl coenzyme A hydroxylase or gamma-butyrobetaine hydroxylase. 2OGdependent oxygenases may be mammalian, preferably human polypeptides.

Human 2OG oxygenases for which it may be desirable not to inhibit whenmodulating HIF asparagine hydroxylase activity include AlkB, collagenprolyl hydroxylases, lysine hydroxylases, the aspartyl/asparaginehydroxylase known to hydroxylate endothelial growth factor domains,phytanoyl CoA hydroxylase, gamma-butyrobetaine hydroxylase, trimethyllysine hydroxylase, HIF prolyl hydroxylase isoforms including PHD1,PHD2, PHD3, and enzymes closely related to FIH including those proteinsin the SWALL database that are referenced by the following numbers:Q9NWJ5, Q8TB10, Q9Y4E2, O95712, Q9H8B1, Q9NWT6 in Homo sapiens, andQ91W88 and Q9ER15 in Mus musculus and homologues of these enzymes. It isalso recognised that in some circumstances it may be advantageous toinhibit FIH and one or more of the aforementioned enzymes, in particularone or more of the HIF prolyl hydroxylase isoforms. Further, ininhibiting some of the above enzymes it may be advantageous not toinhibit FIH and the methods can be used in a method for discovering PHDinhibitors that are not inhibitors of FIH.

The invention provides for the use of such selective inhibitors of HIFasparagine hydroxylases in the manufacture of a medicament for thetreatment of a condition associated with reduced HIF activity.

In alternative aspects of the present invention, the assays can be usedto establish whether agents which have been identified as inhibitors oractivators of other 2OG dependent oxygenases are specific for suchoxygenases, or at least do not affect HIF asparagine hydroxylase. Inparticular the effect of agents which act on the activity of HIFasparagine hydroxylase while not affecting the related HIF prolylhydroxylase, or vice versa, can be established. Thus, the assays may becarried out using agents or modifications of agents which have beenidentified as inhibitors of a 2OG dependent oxygenase, such as collagenprolyl hydroxylase to identify whether such an agent is specific forcollagen prolyl hydroxylase and is not active or shows reduced activityagainst HIF hydroxylases and in particular their asparagine hydroxylaseactivity.

Therapeutic Appplications

A compound, substance or agent which is found to have the ability toaffect the hydroxylase activity of a HIF asparagine hydroxylase, hastherapeutic and other potential in a number of contexts, as discussed.For therapeutic treatment, such a compound may be used in combinationwith any other active substance, e.g. for anti-tumour therapy anotheranti-tumour compound or therapy, such as radiotherapy or chemotherapy.

An agent identified using one or more primary screens (e.g. in acell-free system) as having ability to modulate the HIFα asparaginehydroxylation activity of a HIF hydroxylase may be assessed furtherusing one or more secondary screens. A secondary screen may involvetesting for an increase or decrease in the amount of HIF-α or HIFactivity, for example as manifest by the level of a HIF target gene orprocess present in a cell in the presence of the agent relative to theabsence of the agent.

A HIF hydroxylase or a HIF polypeptide may be used in therapies whichinclude treatment with full length polypeptides or fragments thereof, orotherwise modified polypeptides (e.g. to enhance stability or ensuretargeting, including in conjunction with other active agents such asantibodies. For example, mutation of HIF-1α to replace Asn 803 withanother amino acid residue may prevent hydroxylation and thus promoteinteraction of HIF-α with p300 and stimulate transcriptional activation.

Generally, an agent, compound or substance which is a modulatoraccording to the present invention is provided in an isolated and/orpurified form, i.e. substantially pure. This may include being in acomposition where it represents at least about 90% active ingredient,more preferably at least about 95%, more preferably at least about 98%.Any such composition may, however, include inert carrier materials orother pharmaceutically and physiologically acceptable excipients, suchas those required for correct delivery, release and/or stabilization ofthe active agent. As noted below, a composition according to the presentinvention may include in addition to an modulator compound as disclosed,one or more other molecules of therapeutic use, such as an anti-tumouragent.

Products Obtained by Assays of the Invention

The invention further provides compounds obtained by assay methods ofthe present invention, and compositions comprising said compounds, suchas pharmaceutical compositions wherein the compound is in a mixture witha pharmaceutically acceptable carrier or diluent. The carrier may beliquid, e.g. saline, ethanol, glycerol and mixtures thereof, or solid,e.g. in the form of a tablet, or in a semi-solid form such as a gelformulated as a depot formulation or in a transdermally administerablevehicle, such as a transdermal patch.

The invention further provides a method of treatment which includesadministering to a patient an agent which interferes with thehydroxylation of the asparagine target residue of an HIFα polypeptide bya HIF hydroxylase. Such agents may include inhibitors of asparaginehydroxylase activity.

The therapeutic/prophylactic purpose may be related to the treatment ofa condition associated with reduced or suboptimal or increased HIFlevels or activity, or conditions in which have normal HIP levels, butwhere an modulation in HIF activity such as an increase or decrease inHIF activity is desirable such as:

-   (i) ischaemic conditions, for example organ ischaemia, including    coronary, cerebrovascular and peripheral vascular insufficiency. The    therapy may be applied in two ways; following declared tissue    damage, e.g. myocardial infarction (in order to limit tissue    damage), or prophylactically to prevent ischaemia, e.g. promotion of    coronary collaterals in the treatment of angina.-   (ii) wound healing and organ regeneration-   (iii) auto-, allo-, and xeno-transplantation.-   (iv) systemic blood pressure-   (v) cancer; HIFα is commonly up-regulated in tumour cells and has    major effects on tumour growth and angiogenesis.-   (vi) inflammatory disorders.-   (vii) pulmonary arterial blood pressure, neurodegenerative disease.-   (viii) diabetes    Modulating HIF asparaginyl hydroxylase activity in a person, an    organ, or a group of cells may be exploited in different ways to    obtain a therapeutic benefit.-   (a) Non cell autonomous: The HIF system is used by cells to    influence the production of substances which signal to other cells.    These signals may then have effects at (i) a distant site (for    example erythropoietin acts on the bone marrow) or (ii) locally    (angiogenic growth factors increase the local formation of blood    vessels). Manipulating non cell-autonomous behaviour via altering    hydroxylase activity is therefore useful in the treatment of    anaemia, and local ischaemia, for example in the eye, brain, heart    and limbs. Many other signals that are involved in aspects of    physiological homeostatis may be, or are known to be, adjusted by    HIF activation. Consequently, altering HIF asparaginyl hydroxylase    activity may be used to potentiate or initiate a helpful response    for a therapeutic benefit, or to prevent or ameliorate a harmful    response. For example, this approach can be used to alter appetite,    or blood pressure in the systemic or pulmonary beds.-   (b) Cell autonomous: the HIF system is also used by cells to    regulate cellular metabolism, and decisions concerning    differentiation, proliferation and apoptosis. Therefore manipulating    the HIF system can be used to alter the viability and behaviour of    cells. An increase cell viability can be achieved by increasing HIF    activation, for example in an ischaemic tissue. This approach can    also be used in improving pancreatic beta cell viability as a way of    ameliorating diabetes, or of improving the viability or function of    a group or groups of neurons in Parkinson's disease, motorneurone    disease or forms of dementia. In a different approach, the HIF    signal can be manipulated to prevent a group of cells proliferating,    or to promote its death or differentiation. For example transient    activation of the HIF system in a malignant tumour can be used to    provoke death of a substantial number of tumour cells.    Pharmaceutical Compositions

In various further aspects, the present invention thus provides apharmaceutical composition, medicament, drug or other composition forsuch a purpose, the composition comprising one or more agents, compoundsor substances as described herein, including HIF asparagine hydroxylaseinhibitors, the use of such an composition in a method of medicaltreatment, a method comprising administration of such a composition to apatient, e.g. for treatment (which may include preventative treatment)of a medical condition as described above, use of such an agent compoundor substance in the manufacture of a composition, medicament or drug foradministration for any such purpose, e.g. for treatment of a conditionas described herein, and a method of making a pharmaceutical compositioncomprising admixing such an agent, compound or substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

In one embodiment the method for providing a pharmaceutical compositionmay typically comprise:

-   -   (a) identifying an agent by an assay method of the invention;        and    -   (b) formulating the agent thus identified with a        pharmaceutically acceptable excipient.

The pharmaceutical compositions of the invention may comprise an agent,polypeptide, polynucleotide, vector or antibody according to theinvention and a pharmaceutically acceptable excipient.

The agent may be used as sole active agent or in combination with oneanother or with any other active substance, e.g. for anti-tumour therapyanother anti-tumour compound or therapy, such as radiotherapy orchemotherapy.

Whatever the agent used in a method of medical treatment of the presentinvention, administration is preferably in a “prophylactically effectiveamount” or a “therapeutically effective amount” (as the case may be,although prophylaxis may be considered therapy), this being sufficientto show benefit to the individual. The actual amount administered, andrate and time-course of administration, will depend on the nature andseverity of what is being treated. Prescription of treatment, e.g.decisions on dosage etc, is within the responsibility of generalpractitioners and other medical doctors.

An agent or composition may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated, e.g. as described above.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. In particular they may include a pharmaceutically acceptableexcipient. Such materials should be non-toxic and should not interferewith the efficacy of the active ingredient. The precise nature of thecarrier or other material will depend on the route of administration,which may be oral, or by injection, e.g. cutaneous, subcutaneous orintravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Liposomes, particularly cationic liposomes, may be used in carrierformulations. Examples of techniques and protocols mentioned above canbe found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.(ed), 1980.

The substance or composition may be administered in a localised mannerto a particular site or may be delivered in a manner in which it targetsparticular cells or tissues, for example using intra-arterial stentbased delivery.

Targeting therapies may be used to deliver the active substance morespecifically to certain types of cell, by the use of targeting systemssuch as antibody or cell specific ligands. Targeting may be desirablefor a variety of reasons, for example if the agent is unacceptablytoxic, or if it would otherwise require too high a dosage, or if itwould not otherwise be able to enter the target cells.

In a further embodiment the invention provides for the use of an agentof the invention in the manufacture of a medicament for the treatment ofa condition associated with increased or decreased HIF levels oractivity. The condition may, for example, be selected from the groupconsisting of ischaemia, wound healing, auto-, allo-, andxeno-transplantation, systemic high blood pressure, cancer, andinflammatory disorders.

Gene Therapy

The HIF asparagine hydroxylases of the present invention can be used topromote or enhance hydroxylation of HIF-α in target cells. Suchpromotion of hydroxylation may therefor reduce interaction with p300 andreduce transcription activation. This will be of assistance in reducingangeogenesis and effect other apoptotic and proliferative responses intarget cells. Thus, in accordance with this aspect of the invention anucleic acid encoding a HIF asparagine hydroxylase may be provided totarget cells in need thereof.

Where the substances are peptides or polypeptides, they may be producedin the target cells by expression from an encoding nucleic acidintroduced into the cells, e.g. from a viral vector. The vector may betargeted to the specific cells to be treated, or it may containregulatory elements which are switched on more or less selectively bythe target cells.

Nucleic acid encoding a substance e.g. a peptide able to modulate, e.g.interfere with, asparagine hydroxylation of HIFα by a HIF hydroxylase,may be used in methods of gene therapy, for instance in treatment ofindividuals, e.g. with the aim of preventing or curing (wholly orpartially) a disorder.

Nucleic acid encoding a HIF asparagine hydroxylase as described hereinmay also be used in the anti-sense regulation of the HIF hydroxylaseactivity. Down-regulation of expression of a gene encoding a HIFhydroxylase may be achieved using anti-sense technology, or RNAinterference.

In using anti-sense genes or partial gene sequences to down-regulategene expression, a nucleotide sequence is placed under the control of apromoter in a “reverse orientation” such that transcription yields RNAwhich is complementary to normal mRNA transcribed from the “sense”strand of the target gene. See, for example, Smith et al, (1988) Nature334, 724-726. Antisense technology is also reviewed in Flavell, (1994)PNAS USA 91, 3490-3496.

The complete sequence corresponding to the reverse orientation of thecoding sequence need not be used. For example, fragments of sufficientlength may be used. It is a routine matter for the person skilled in theart to screen fragments of various sizes and from various parts of thecoding sequence to optimise the level of anti-sense inhibition. It maybe advantageous to include the initiating methionie ATG codon, andperhaps one or more nucleotides upstream of the initiating codon. Afurther possibility is to target a conserved sequence of a gene, e.g. asequence that is characteristic of one or more genes, such as aregulatory sequence.

The sequence employed may be 500 nucleotides or less, possibly about 400nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100nucleotides. It may be possible to use oligonucleotides of much shorterlengths, 14-23 nucleotides, although longer fragments, and generallyeven longer than 500 nucleotides are preferable where possible.

Anti-sense oligonucleotides may be designed to hybridise to thecomplementary sequence of nucleic acid, pre-mRNA or mature mRNA,interfering with the production of a HIF hydroxylase encoded by a givenDNA sequence (e.g. either native polypeptide or a mutant form thereof),so that its expression is reduce or prevented altogether. Anti-sensetechniques may be used to target a coding sequence; a control sequenceof a gene, e.g. in the 5′ flanking sequence, whereby the anti-senseoligonucleotides can interfere with control sequences. Anti-senseoligonucleotides may be DNA or RNA and may be of around 14-23nucleotides, particularly around 15-18 nucleotides, in length. Theconstruction of antisense sequences and their use is described in Peymanand Ulman, Chemical Reviews, 90:543-584, (1990), and Crooke, Ann. Rev.Pharmacol. Toxicol., 32:329-376, (1992).

It may be preferable that there is complete sequence identity in thesequence used for down-regulation of expression of a target sequence,and the target sequence, though total complementarity or similarity ofsequence is not essential. One or more nucleotides may differ in thesequence used from the target gene. Thus, a sequence employed in adown-regulation of gene expression in accordance with the presentinvention may be a wild-type sequence (e.g. gene) selected from thoseavailable, or a mutant, derivative, variant or allele, by way ofinsertion, addition, deletion or substitution of one or morenucleotides, of such a sequence.

The sequence need not include an open reading frame or specify an RNAthat would be translatable. It may be preferred for there to besufficient homology for the respective sense RNA molecules to hybridise.There may be down regulation of gene expression even where there isabout 5%, 10%, 15% or 20% or more mismatch between the sequence used andthe target gene.

Other approaches to specific down-regulation of genes which may be usedto modulate HIF asparagine hydroxylase expression are well known,including the use of ribozymes designed to cleave specific nucleic acidsequences. Ribozymes are nucleic acid molecules, actually RNA, whichspecifically cleave single-stranded RNA, such as mRNA, at definedsequences, and their specificity can be engineered. Hammerhead ribozymesmay be preferred because they recognise base sequences of about 11-18bases in length, and so have greater specificity than ribozymes of theTetrahymena type which recognise sequences of about 4 bases in length,though the latter type of ribozymes are useful in certain circumstances.References on the use of ribozymes include Marschall, et al. Cellularand Molecular Neurobiology, 1994. 14(5): 523; Hasselhoff, Nature 334:585 (1988) and Cech, J. Amer. Med. Assn., 260: 3030 (1988).

Vectors such as viral vectors have been used in the prior art tointroduce nucleic acid into a wide variety of different target cells.Typically the vectors are exposed to the target cells so thattransfection can take place in a sufficient proportion of the cells toprovide a useful therapeutic or prophylactic effect from the expressionof the desired peptide. The transfected nucleic acid may be permanentlyincorporated into the genome of each of the targeted cells, providinglong lasting effect, or alternatively the treatment may have to berepeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are knownin the art, see U.S. Pat. No. 5,252,479 and WO93/07282. In particular, anumber of viruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpesviruses, includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have used disabled murine retroviruses.

As an alternative to the use of viral vectors in gene therapy otherknown methods of introducing nucleic acid into cells includes mechanicaltechniques such as microinjection, transfer mediated by liposomes andreceptor-mediated DNA transfer.

Receptor-mediated gene transfer, in which the nucleic acid is linked toa protein ligand via polylysine, with the ligand being specific for areceptor present on the surface of the target cells, is an example of atechnique for specifically targeting nucleic acid to particular cells.

In various further aspects, the present invention thus provides apharmaceutical composition, medicament, drug or other composition foruse in a method of treating a medical condition described above, thecomposition comprising an isolated nucleic acid molecule as describedherein, the use of such an composition in a method of medical treatment,a method comprising administration of such a composition to a patient,e.g. for treatment (which may include preventative treatment) of amedical condition as described above, use of such an agent compound orsubstance in the manufacture of a composition, medicament or drug foradministration for any such purpose, e.g. for treatment of a conditionas described herein, and a method of making a pharmaceutical compositioncomprising admixing such an agent, compound or substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

Use of Polypeptides

Another aspect of the present invention provides the use of a HIFhydroxylase as described herein or a fragment thereof for the asparaginehydroxylation of an HIF polypeptide, or an asparagine-containingsubstrate of HIF hydroxylase.

In particular, the present inventions have established that FIHhydroxylates an asparagine residue in HIF-α, but does not appear to haveactivity in hydroxylating an aspartic acid residue. Accordingly,polypeptides of the invention, that is SEQ ID NO: 2 and variants thereofas described above, may be used to specifically hydroxylate asparagineresidues in a substrate with little or no hydroxylation of asparticacid.

A HIF asparagine hydroxylase polypeptide according to the presentinvention can also be used to identify additional substrates of HIFhydroxylases. For example, peptides which have either previously beendemonstrated to be hydroxylated by other hydroxylases, or other peptidesmay be brought into contact with a HIF hydroxylase according to thepresent invention and monitoring for asparagine hydroxylation of suchpeptides. Any suitable conditions may be selected including theprovision of agents and co-factors known to enhance hydroxylation by thehydroxylases of the present invention. Hydroxylation of the substratemay be monitored by any suitable method including monitoring levels ofco-factors or by products of hydroxylation.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures described below.

DESCRIPTION OF THE FIGURES

FIG. 1 a: Electrospray ionisation mass spectra of HIF-1α 775-826demonstrating the incorporation of oxygen from dioxygen into product: a)HIF-1α 775-826 before reaction, calculated mass 6697.31 Da, observedmass (MH+)=6694.7 Da; b) peptide isolated from reaction under norm-oxic(¹⁶O₂) conditions, mass increase=16.5 Da; c) peptide isolated fromreaction under ¹⁸O₂ atmosphere, mass increase=18.3 Da.

FIG. 1 b: FIH modulates HIF-1α CAD binding to the CH1 domain of p300.The autoradiograph shows capture of ³⁵S-labeled CH1 by HIF-1α CAD thathad or had not been pre-incubated with recombinant FIH. Pre-incubationwith FIH strikingly reduced ability of HIF-1α CAD to capture CH1(compare lanes 1 and 2). Inclusion of N-oxaloylglycine in the FIH/HIF-1αCAD reaction step inhibited the effect of FIH. Binding of CH1 to N803Amutant HIF-1α CAD that is constitutively active was unaffected bypre-incubation with FIH (lanes 4 and 5).

FIG. 2 a: Sequence alignment of CAS1 (clavaminic acid synthase 1,Q05581); PHD3 (prolyl hydroxylase domain containing protein 3, Q9H6Z9);FIH1 (factor inhibiting HIF-1, Q969Q7); PMI (phosphomannose isomeraseType II, P34948), comparing conserved secondary structure and bindingmotifs. The crystallographically assigned secondary structure of CAS1 isindicated above the alignment and PMI below the alignment, jellyrollβ-strands are numbered. The α-helices and β-strands not in the jellyrollcan also be seen. Note the common presence of an insert betweenβ-strands 4 and 5 of the jellyroll in FIH, also present in CAS 1 andtaurine dioxygenase (not shown), which define a subfamily of 2-OGdioxygenases. The known/predicted metal-binding ligands are indicated bya triangle. In reported enzyme:Fe(II):2-OG structures the 5-carboxylateof 2-OG forms an electrostatic interaction with the side-chain of anarginine residue (indicated by a triangle). FIH does not possess eitherof the RX₃T (as in the PHDs) or RXS motifs identified as binding the2-OG 5-carboxylate, nor a lysine in an analogous position to thearginine of these motifs (as in procollagen prolyl hydroxylase). In FIH,Thr-290 and Thr-292, located on the 8^(th) jellyroll β-strand, may beinvolved in 2-OG binding.

FIG. 2 b: Comparison of structures of phosphomannose isomerase complexedwith zinc (left) and clavaminic acid synthase complexed with Fe(II),2-OG and (L)-N-α-acetylarginine substrate (right). The core jellyrollmotif, the insert to this jellyroll motif and other secondary structurecan be seen. The analogously located metal-binding residues and the twosubstrates of CAS1 can also be seen. Note PMI possess an extended insert(˜115 residues) to the jellyroll motif as in CAS1 and predicted for FIH.

EXAMPLES Experimental Procedures

Cloning, expression and purification of fih/FIH—The fih gene was PCRamplified from Image clone 4138066 and subcloned as an Nde I/BamH Ifragment into the pET28a(+) vector (Novagen) in order to generate anN-termilal His₆-tagged FIH fusion protein. Primers used were: forward;5′-gggaattccatatggcggcgacagcggcg-3′(SEQ ID NO:4), reverse;5′cgcggatccctagttgtatcggccc-3′(SEQ ID NO:5). Following amplification theintegrity of fih was confirmed by DNA sequencing. The fih/pET28a(+)construct was transformed into Escherichia coli BL21(DE3) and grown at37° C. in 2TY media containing 30 μg/ml kanamycin and was induced withisopropyl-1-(D)-thiogalactoside (IPTG). The N-terminal His₆-tagged FIHfusion protein was purified using nickel affinity chromatography(Novagen) and the N-terminal His₆ tag removed from FIH by cleavage withthrombin. Size exclusion chromatography (Superdex S75) yielded FIHof >95% purity by SDS-PAGE analysis. Electrospray ionisation MS revealedthe mass of the isolated FIH was consistent with that expected from itspredicted amino acid sequence (observed 40,556 Da, calculated 40,567Da).

Cloning expression and purification of HIF-1α fragments—The desiredsegments of Human HIF-1α were cloned directly as Sac II/Asc I fragmentsfrom pcDNA3 vectors (O Rourke et al (1999) J. Biol Chem 274, 2060-2071)into a modified version of pGEX-6P-1 (Amersham Biosciences) in order togenerate N-terminal glutathione S-transferase (GST) tagged fusionproteins. The constructs were transformed into E. coli BL21(DE3) andgrown at 37° C. in 2TY media containing 100 μg/ml ampicillin and wereinduced with IPTG. The GST-tagged fusion proteins were purified usingGlutathione Sepharose™ 4B resin (Amersham Biosciences). The GST tag wasremoved from the HIF-1α fragments when required by PreScission™ Proteasetreatment. Size exclusion chromatography (Superdex S75) yielded >95%pure protein by SDS-PAGE analysis. His₆-tagged sections of HIF-1α wereprepared as described in Epstein et al, 2001. Cell, 107 43-54.

Mutagenesis studies of HIF-1α 775-826—Mutant GST HIF-1α 775-826isoenzymes were produced from the wild-type construct using theQuikchange system (Stratagene). The primers for each of the asparaginemutations were: N803A;

forward: (SEQ ID NO:6) 5′-gttatgattgtgaagttgctgctccatatacaaggc-3′,reverse: (SEQ ID NO:7) 5′-gtataggagcagcaacttcacaatcataactgg-3′ N803Q;forward: (SEQ ID NO:8) 5′-gttatgattgtgaagttcaagctcctatacaaggc-3′reverse: (SEQ ID NO:9) 5′-gtataggacgttgaacttcacaatcataactgg-3′, N803E;forward: (SEQ ID NO:10) 5′-gttatgattgtgaagttgaagctcctatacaaggc-3′reverse: (SEQ ID NO:11) 5′-ttgtataggagcttcaacttcacaatcataactgg-3′,N803D; forward: (SEQ ID NO:12) gttatgattgtgaagttgatgctcctatacaaggc-3′,reverse; (SEQ ID NO:13) 5′-ttgtataggagcatcaacttcacaatcataactgg.Mutations were confirmed by DNA sequencing.

FIH assays—Assays for decarboxylation of 2-OG were performed usingradiolabelled 2-OG (New England Nuclear) as reported in Epstein et alsupra. Incubations contained ascorbate, DTT, catalase, 2-OG, FIH, andsubstrate in 50 mM Tris/HCl pH7.4, and were carried out at 37° C.typically for 20 minutes. HPLC separations were achieved on a PhenomenexJupiter C4 (10 cm×4.6 mm) column by using of a linear gradient of CH₃CNin 0.1% TFA. For inhibition and metal dependence assays, GST HIF-1α775-826 was used as substrate at 30 μM, and with iron(II) ammoniumsulfate, cobalt(II) chloride, zinc(II) chloride, and N-oxaloylglycine atfinal concentrations of 0, 2, 10, 40, 80 and 200 μM. Assays under anatmosphere of ¹⁸O₂ were performed as reported in McNeill et al, BioorgMed Chem Lett, 2002 12, 1547-1550.

GST pulldown assays—To examine for effects of FIH on HIF-1α binding top300, the GST fusion protein expressing amino acids of HIF-1α 775-826and the N803A mutant were exposed to recombinant FIH under conditionsdesigned to promote the hydroxylation. N-oxaloylglycine was added to 5mM when required. Glutathione Sepharose beads were added for 40 minutesat 25° C. and then washed with 50 mM Tris/HCl pH 7.4, 0.5% NP40, 150 mMNaCl and 1 mM DTT. The beads bearing the treated GST HIF fusion proteinswere mixed with ³⁵S labeled CH1-p300 (pcDNA3 Gal-p300-CH1 encoding aminoacids 300-528 of p300) produced in rabbit reticulocyte lysate. Bindingwas performed for 1 hour at 25° C. and the beads were then-washed in thesame buffer. The bound proteins were eluted in SDS running buffer andanalyzed by SDS-PAGE and autoradiography.

Fluorescence polarisation assay—Assays for hydroxylation of HIFpolypeptide can be carried out in 50 mM Tris/HCl pH7.5. Incubationscontain 4 mM ascorbic acid, 1 mM DTT, 80 microM 2OG, polypeptide, 468 nMGST-p300 complex (where GST is glutathion S transferase, a purificationaid), 15 microM iron(II), and 0.3 nM HIF hydroxylase. Incubation may beat 29° Celsius. A graph can be plotted of data in real time from theincubation of a HIF hydroxylase with a labelled (Cy5 fluorescence dye)HIF fragment in the presence of p300 to which hydroxylated peptide bindsresulting in an increase in anisotropy.

FIH is a HIF-α CAD hydroxylase—Preliminary analysis of the predicted FIHamino acid sequence in light of the sequences for 2-OG oxygenases ofknown structure and function suggested that FIH contained the conservedHX(D/E) . . .H ‘facial triad’ of residues that binds the Fe(II)cofactor. Given the reported interaction of FIH with the C-terminalactivation domain of HIF-1α and its ability to downregulatetransactivation we considered that FIH might function as the Asn-803hydroxylase.

N-Terminally His₆-tagged FIH gene was therefore produced in E. coliBL21(DE3) and purified to >95% purity. HIF-1α polypeptides encompassingall three identified sites of hydroxylation were then examined asputative substrates for purified recombinant FIH (Table 1, entries 1-6),using an assay that monitors decarboxylation of 2-OG. The resultsclearly indicate that there was no FIH mediated hydroxylation of thoseHIF fragments previously shown to be substrates for the PHD isozymesthereby implying that FIH is not a HIF prolyl hydroxylase (Table 1,entries 4-6). In contrast, CO₂ production was strikingly stimulated inthe presence of HIF-1α fragments containing the C-terminal activationdomain (CAD, Table 1, entries 1-3). Both GST-fused and free forms of apolypeptide encompassing the human HIF-1α CAD (residues 775-826) wereobserved to cause significant stimulation of FIH mediated 2-OG turnover.A high ratio (>10:1) of prime substrate coupled:uncoupled 2-OG turnoverwas observed in the case of the free 775-826 substrate, and preliminarykinetic parameters for this peptide obtained using the 2-OG turnoverassay were K_(m)(peptide)=10 μM, K_(m)(2-OG)=10 μM, V_(max)=0.3μmol/min/mg.

TABLE 1 Substrate selectivity of FIH with HIF-1α polypeptides. FIH wasincubated with proteins containing the three known hydroxylation sites(Pro-402, Pro-564, Asn-803) in HIF-1α. Column 3 indicates activity ofpolypeptides as assayed by CO₂ release under the standard assayconditions. Column 4 shows the activity of each substrate relative tothe activity of 775-826 peptide as a substrate under the sameconditions. See Experimental for assay details. CO₂ released % activityrelative Entry Substrate (nmol) to 775-826 peptide 1 HIF-1α 775-826 2.8100 2 GST HIF-1α 775-826 2.7 96 3 GST HIF-1α 577-826 0.81 67 4 GSTHIF-1α 530-652 0.05 1.8 5 His₆-HIF-1α 344-503 <0.001 <0.01 6 His₆-HIF-1α530-698 <0.001 <0.01 7 GST HIF-1α 775-826 N803D 0.22 7 8 GST HIF-1α775-826 N803A <0.001 <0.01 9 GST HIF-1α 775-826 N803E <0.001 <0.01 10GST HIF-1α 775-826 N803Q <0.001 <0.01

Since the 2-OG turnover assay does not directly measure oxidation of theprime substrate, unequivocal demonstration of FIH mediated hydroxylationwas sought from mass spectrometric analyses. Using HIF-1α 775-826peptide as a substrate under normoxic conditions, an increase in mass of16Da was observed after HPLC isolation of product (FIG. 1 a, panels aand b). The results demonstrate that FIH is a 2-OG dependent hydroxylasethat modifies the HIF-1α CAD. To test whether this activity regulatesthe interaction with the p300 co-activator, GST-fused HIF-1α CAD wasexposed to purified recombinant FIH under conditions that supportdecarboxylation of 2-OG, purified, and assayed for the ability tointeract with ³⁵S-labelled CH1 by GST pulldown assay. FIG. 1 b showsthat pre-treatment with FIH greatly reduced the ability of the HIF-1αCAD polypeptide to interact with CH1 (compare lanes 1 and 2).

Following previous MS/MS assignment of Asn-803 as the modified residuein the HIF-1α CAD, we constructed a series a mutations at this site andperformed further 2-OG decarboxylation and interaction assays using themutant polypeptides. Mutation of Asn-803 in GST HIF-1α 775-826 toalanine abolished all activity in the 2-OG decarboxylation assay (Table1, entries 8-10), and prevented modulation of CH1 binding in theinteraction assay (FIG. 1 b, lanes 4 and 5). Mutation to glutamine andglutamate also abolished activity, whilst an Asp-803 mutant stillsupported some 2-OG turnover, but only at a maximum of 7% of theanalogous Asn-803 substrate (Table 1, entry 7).

Taken together these results demonstrate that FIH is the dioxygenrequiring Asn-803 hydroxylase that controls HIF-α C-terminaltransactivation by regulating the interaction with the CH1 domain ofp300. The clear preference of FIH for an asparaginyl rather than anaspartyl residue as substrate contrasts with the previously reportedhuman Asp/Asn hydroxylase which catalyses hydroxylation at the β-carbonof both aspartyl and asparaginyl residues. This observation raises thepossibility that FIH mediated hydroxylation does not occur at theβ-carbon of Asn-803 but at another atom. Oxidation of either theα-carbon or the carbonyl oxygen of the primary amide seems unlikelygiven the relative lack of activity of the Asp-803 and other mutants andlikely instability of the putative products. It is more likely thatnitrogen of the primary amide is hydroxylated to give a hydroxamic acid(CH₂CONHOH). The available NMR data suggests that such a modificationwould be disruptive to the interaction between CAD and p300.

Further characterization of FIH activity—To explore the mechanism of FIHand its relationship to the characteristics of in vivo HIF activation,in vitro analyses of FIH using the purified recombinant protein wereperformed. Analyses revealed that the purified recombinant FIH containedca. 1 metal ion per protein, consistent with the operation of amono-iron catalytic site (unpublished data). FIH was inhibited byN-oxaloylglycine, a known inhibitor of the PHD isozymes and other 2-OGoxygenases, both in bioassays of CH1 capture by HIF-1α CAD (FIG. 1 bcompare lanes 2 and 3) and in vitro kinetic assays (IC₅₀, 25 μM). Cobalt(II) also inhibited recombinant FIH activity (IC₅₀, 10 μM), explainingthe ability of these substances to regulate HIF-1α CAD activity.Interestingly addition of exogenous iron (II) to the purified FIH/HIF-1αCAD assay produced no further increase in activity, implying relativelytight metal binding by FIH. This may reflect the ability of thehydroxylated asparagine product to chelate iron. Because of therelationship of FIH to the zinc (II) binding proteins from the cupinfamily (see below) we also tested the action of zinc (II) and foundsimilar inhibition to that with cobalt (II) (IC₅₀, 10 μM). Though zinc(II) does not induce a HIF transcriptional response, recent studies havedemonstrated that it does stabilize HIF-1α, but blocks transactivationby inducing alternative splicing to a shortened form that lacks the CAD.Though the physiological relevance of these findings is still unclear ittherefore seems likely that zinc (II) inhibits both FIH and the PHDenzymes.

The requirement of FIH for dioxygen as a co-substrate indicates that,like the PHD isozymes, FIH may act as a cellular oxygen sensor. Indeed,when assayed under conditions of graded reduction of atmospheric oxygen,FIH demonstrated large and progressive reductions in HIF-1α CAD linked2-OG decarboxylation below ca. 5% O₂. This suggests that, as for PHD1,activity is limited by oxygen concentrations in the physiological range.To explore further the interaction with dioxygen, the origin of theincorporated oxygen atom in the product was investigated by incubationunder an atmosphere of ¹⁸O₂ gas followed by mass spectrometric analysis.The results reveal the source of the oxygen in the hydroxylated productto be >98% derived from dioxygen (FIG. 1 a, panel c), demonstrating adirect interaction between FIH and dioxygen, as shown for PHD1 andprocollagen prolyl hydroxylase. These results contrast with similarincorporation experiments with microbial 2-OG oxygenases whereincorporation of oxygen from water as well as dioxygen is observed. Theexchange process has been proposed to occur via binding of water to apentacoordinate ferryl species[Fe(IV)=O<->Fe(III)-O]. In the case of FIHand PHD1, it may be that the presence of a large peptide substrate atthe active site blocks access of water so preventing exchange.

Biological and structural implications—The involvement of a furthermember of the 2-OG oxygenase superfamily, distinct from the PHDisozymes, in the regulation of HIF transactivation raises severalbiological issues. First it defines another link between theavailability of dioxygen and HIF activity that may help shape thephysiological characteristics of the transcriptional response tohypoxia. Second it provides an explanation for the characteristic actionof cobalt in mimicking the effect of hypoxia on both the isolateddegradation and activation domains of HIF-α subunits. Third it providesa further target for the development of therapeutic agents that augmentHIF activity in ischaemia/hypoxic disease. Fourth the reportedinteractions of FIH with both histone deacetylases and pVHL suggeststhat these proteins may be involved in additional oxygen regulatedprocesses that affect the HIF transcriptional response or otherpathways.

Our findings also raise interesting structural and evolutionary issueswith respect to the 2-OG oxygenases and related enzymes. Sequencecomparisons reveal that FIH, like the PHD isozymes, employs a2-His-1-carboxylate facial triad formed from a conserved HX(D/E) . . . Hmotif, as found in other 2-OG oxygenases. Together with crystallographicinsights and secondary structure predictions, the analyses imply thatFIH possesses the jellyroll β-sheet core (double stranded β-helix)common to other 2-OG oxygenases. They also reveal that FIH possesses aca. 40 residue insert between β-strands 4 and 5 of the 8-strandedjellyroll motif (FIG. 2 a) and suggest that the structure of the FIHwill be significantly different to the PHD isozymes, which are closelyrelated to one another.

Though kinetic analyses clearly demonstrate a requirement of FIH for2-OG, an unusual feature of FIH concerns the identity of residuesinvolved in binding the 5-carboxylate of 2-OG. The analyses imply thatFIH does not possess an arginine or lysine residue, located on β-strand8 of the jellyroll, that is involved in binding the 2-OG 5-carboxylatepresent in the PHDs and many other 2-OG oxygenases (FIG. 2 a).

Thus, FIH probably constitutes the first member of a new structuralsubfamily of 2-OG oxygenases and the development of inhibitors selectivefor FIH versus the PHD isozymes or vice versa should be possible.

Mahon et al. used BLAST searches to identify proteins with sequencesimilarity to FIH. One of these (Genbank AF 168362.1) is a JmjC homologyregion, present in the jumonji transcription factors, which have beenidentified as members of the cupin structural superfamily. Theidentification of FIH as an Fe(H) dependent oxygenase inhibited byZn(II) prompted us to compare crystal structures from the 2-OG and cupinsuperfamilies. These analyses revealed a striking similarity between thecores of the 2-OG oxygenases, exemplified by clavaminic acid synthase(CAS1²), and the cupin superfamily, exemplified by phosphomannoseisomerase (PMI³)(27), which, together with the presence of conservedmotifs, suggests the 2-OG oxygenases belong to the cupin superfamily(FIG. 2 b). Further, the HXH . . . H metal binding motif is wellestablished within the cupin superfamily (e.g. in quercetin2,3-dioxygenase) and is modified to a QXH . . . H motif in the case ofType II PMI. Interestingly, the JmjC transcription factors have beenimplicated in cell growth and heart development, and possess a conservedHX(D/E) . . . H motif as in the 2-OG oxygenases, suggesting that, likeFIH and the PHD isozymes, they might be iron oxygenases involved in theregulation of transcription.

1. A method of identifying an agent which inhibits hydroxylation ofhypoxia inducible factor (HIF), the method comprising: introducing intoa cell that expresses a substrate of HIF asparagine hydroxylase a vectorexpressing HIF asparagine hydroxylase, the HIF asparagine hydroxylasehaving the amino acid sequence of SEQ ID NO: 2, or a variant or fragmentthereof having at least 95% identity to the amino acid sequence of SEQID NO: 2 and having HIF asparagine hydroxylase activity; contacting thecell with a test substance under conditions in which asparagine in thesubstrate is hydroxylated in the absence of the test substance; anddetermining hydroxylation of the substrate, wherein a decrease in thehydroxylation of the asparagine in the substrate in the presence of thetest substance compared with the hydroxylation of the asparagine in thesubstrate in the absence of the test substance identifies the testsubstance as the agent which inhibits hydroxylation of HIF.
 2. Themethod of claim 1, wherein the cell is a yeast cell.
 3. The method ofclaim 1, wherein the substrate is an HIF polypeptide.
 4. The method ofclaim 3, wherein the HIF polypeptide comprises HIF-1α, a fragmentthereof comprising Asn 803 of HIF-1α, or a peptide analogue of HIF-1αorfragment thereof comprising an asparagine equivalent to Asn 803 ofHIF-1α.
 5. The method of claim 4, wherein the substrate comprises theCAD domain of the human HIF-1α polypeptide sequence.
 6. The method ofclaim 4, wherein hydroxylation of Asn 803 or of the equivalentasparagine is determined.
 7. The method of claim 4, whereinhydroxylation of the substrate is determined by monitoring theinteraction of the HIF polypeptide with the CH1 domain of p300.
 8. Themethod of claim 4, wherein hydroxylation of the substrate is determinedby monitoring mediated transcription or expression of a reporter genedriven by a HIF regulated promoter.
 9. The method of claim 4, whereinthe contacting step is conducted in the presence of 2-oxoglutarate ordioxygen.
 10. The method of claim 1, wherein hydroxylation of thesubstrate is determined by monitoring the interaction of the HIFpolypeptide with the CH1 domain of p300.
 11. The method of claim 1,wherein the hydroxylation of the substrate is determined by monitoringHIF mediated transcription or expression of a reporter gene driven by aHIF regulated promoter.
 12. The method of claim 1, further comprisingformulating an agent identified as a modulator of HIF asparaginehydroxylase with a pharmaceutically acceptable excipient.
 13. The methodof claim 1, wherein the contacting step is conducted in the presence of2-oxoglutarate or dioxygen.
 14. The method of claim 1, furthercomprising: contacting the test substance with one or more 2OG-dependentoxygenases under conditions in which the 2OG-dependent oxygenases areactive; and determining the activity of the oxygenases, whereinincreased modulation of a HIF asparagine hydroxylase relative to the oneor more 2OG-dependent oxygenases indicates that the test compound isselective for the HIF asparagine hydroxylase.
 15. A method ofidentifying an agent which promotes hydroxylation of hypoxia induciblefactor (HIF), the method comprising: introducing into a cell thatexpresses a substrate of HIF asparagine hydroxylase a vector expressingHIF asparagine hydroxylase, the HIF asparagine hydroxylase having theamino acid sequence of SEQ ID NO: 2, or a variant or fragment thereofhaving at least 95% identity to the amino acid sequence of SEQ ID NO: 2and having HIF asparagine hydroxylase activity; contacting the cell witha test substance under conditions in which asparagine in the substrateis hydroxylated in the absence of the test substance; and determininghydroxylation of the substrate, wherein an increase in the hydroxylationof the asparagine in the substrate in the presence of the test substancecompared with the hydroxylation of the asparagine in the substrate inthe absence of the test substance identifies the test substance as theagent which promotes hydroxylation of HIF.
 16. A method of identifyingan agent which inhibits hydroxylation of hypoxia inducible factor (HIF),the method comprising: contacting a recombinant HIF asparaginehydroxylase with a test substance in the presence of a substrate of theHIF asparagine hydroxylase under conditions in which asparagine in thesubstrate is hydroxylated in the absence of the test substance, the HIFasparagine hydroxylase having the amino acid sequence of SEQ ID NO: 2,or a variant or fragment thereof having at least 95% identity to theamino acid sequence of SEQ ID NO: 2 and having HIF asparaginehydroxylase activity; and determining hydroxylation of the substrate,wherein a decrease in the hydroxylation of the asparagine in thesubstrate in the presence of the test substance compared with thehydroxylation of the asparagine in the substrate in the absence of thetest substance identifies the test substance as the agent which inhibitshydroxylation of HIF.